The invention relates to compounds for tumor diagnosis that consist of conjugates of dyes with short-chain peptides, which are derived from vasco-active intestinal peptide, somatostatin or neurotensin, the use of these compounds as optical diagnostic agents, and diagnostic agents that contain these compounds.
On a cellular level, disease-induced alterations are often manifested as a receptor distribution or expression that is altered relative to the normal state. These differences can be both of quantitative type (e.g., the amount of transferrin receptors in proliferating cells) or else also of qualitative type (e.g., expression of vascular endothelial growth factors, VEGF). Previous attempts to image a pathological receptor expression or distribution have been noted mainly in radiodiagnosis because of the necessary sensitivity of the detection process.
Heptahelical receptors are target molecules of many pharmacological active ingredients (e.g., xcex2-blockers, H2-acid blockers, antihistamines). In addition to therapeutic batches, mainly radiolabled, agonistic ligands of these receptors are used diagnostically for the so-called receptor scintigraphy for in-vivo detection and location of tumors. In this case, the mechanism of the receptor-mediated endocytosis is used, e.g., by the somatostatin receptor, which is more strongly expressed in neuroendocrine tumors. The somatostatin analog 111In-DTPA-pentetreotide (octreoscan(R)) is clinically approved for routine scintigraphic diagnosis; literature: J. Steroid Biochem. Mol. Biol. 37, 1079-82, 1990, J. Nucl. Med. 32, 1184-9, 1991,; J. Nucl. Med. 33, 652-8, 1992; Digestion 3, 54-9, 1994, J. Clin. Invest. 93, 1321-5, 1994, Metabolism 45, 21-3, 1996.
Another batch consists in the use of radiolabeled VIP and VIP-analogs, which bind to the VIP-receptors. The VIP-receptor is more strongly expressed by a broad spectrum of tumors (i.a., adenocarcinomas).
WO 96/30055 describes radiodiagnostic and radiotherapeutic reagents, special VIP-receptor-binding peptides, which are radiolabeled and can be used for radiodiagnosis and radiotherapy. VIP-receptor-binding peptides that can be labeled with Tc-99m for scintigraphy are described especially advantageously. Additional literature: Cancer Research 54, 690-700, 1994; Endocrinology 136, 2662-80, 1994, J. Nucl. Med. 40, 353-361, 1999.
All described diagnosis batches that are based on the somatostatin receptor and VIP-receptor are radiodiagnostic batches (scintigraphy with 123I, 125I, 111In or 99mTc-labeled peptides).
Literature: EP 588754, U.S. Pat. Nos. 5,650,134; 5,620,675, 5,225,180; WO 96/23527; J. Steroid Biochem. Mol. Biol. 37, 1083-87, 1990; Lancet 242-4, 1989, J. Nucl. Med. 39, 1913-17, 1998.
No fluorescence-labeled peptides that are conjugated with dyes that make possible an in-vivo-fluorescence detection of tumors are known to date, however (Photochem. Photobiol. 68, 603-632, 1998).
The object of the invention is to make available new compounds that make possible a sensitive diagnosis of tumors by detection of fluorescence radiation with use of a receptor-specific binding of the compounds to the target tissue. In this case, special dye molecules that are coupled to biomolecules are to yield a highly sensitive, detectable fluorescence signal.
The object is achieved by the provision of compounds that contain fluorescence dyes, which are coupled covalently to short-chain peptides. These conjugates have a high binding affinity to heptahelical receptors, especially the somatostatlin receptor, the VIP-receptor (vaso-active intestinal peptide), and the neurotensin receptor, and they are optionally taken up intracellularly by receptor-mediated endocytosis. The compounds according to the invention are therefore suitable for the technically simple, harmless optical diagnosis of tumor cells and tumor tissues, which increasingly express somatostatin receptors, VIP-receptors or neurotensin receptors in comparison to healthy cells. Especially suitable are the compounds for fluorescence diagnosis and especially advantageously for the fluorescence-endoscopic diagnosis in hollow organs, such as the esophagus, the cervix, the colon, and the bronchial tubes of various tumor types, such as, e.g. adenocarcinomas, neuroendocrine tumors or ductal pancreatic tumors.
Especially preferred dyes are distinguished in that they satisfy certain photophysical and chemical requirements. From the photophysical standpoint, the dyes must have high absorption coefficients and high fluorescence quantum yields to provide an effective signal even in the case of the smallest tissue concentrations. The absorption maxima must overlap a wide spectral range in a freely selectable manner. Thus, for detection in lower tissue layers (several centimeters below the surface), the spectral range of between 600 and 900 nm is essential, while for surface detection, absorption wavelengths of 400 to 600 nm are sufficient. From the chemical standpoint, the dyes must have a high photostability and must exhibit no signs of decomposition (photobleaching) during excitation. The dyes must be usable as synthesis components in the solid-phase-synthetic production of peptides and thus be stable under common synthesis conditions so that a simple, advantageous production of structurally defined dye-peptide-conjugates with solid stoichiometric ratios is ensured between dye and peptide. The requirements are best satisfied by polymethine dyes, especially cyanine, merocyanine, oxonol and squarilium dyes.
Subjects of the invention are therefore peptide-polymethine dye-conjugates of general formula (I)
A1xe2x80x94(X)mxe2x80x94A2 xe2x80x83xe2x80x83I
in which
X stands for an xcex1-, xcex2- or xcex3-amino acid with D- or L-configuration, and
m stands for a number from 5 to 30, whereby the resulting amino acid sequence (X)m can be cyclized in a straight-chain nature or via a disulfide bridge between two cysteines or homocysteines or amidically between the N- and C-terminus and stands for the amino acid sequence of the vaso-active intestinal peptide (VIP), the somatostatin or the neurotensin, or for fragments, partial sequences, derivatives or analogs of the VIP, somatostatin or neurotensin,
A1 stands for a hydrogen atom, an acetyl radical or an alkyl radical with up to 10 C atoms, which optionally can be substituted with 1 to 3 carboxy groups and/or 1 to 6 hydroxy groups, or a poly(oxyethylene) radical with 2 to 30 xe2x80x94CH2CH2O units, or a dye molecule from the class of the polymethine dyes, which has at least one absorption maximum in the range of 380 to 1200 nm,
A2 stands for a hydroxy group, an amino group or a dye molecule from the class of polymethine dyes, which has at least one absorption maximum in the range of 380 to 1200 nm, under the condition that at least one of radicals A1 or A2 represents a dye molecule from the class of polymethine dyes, which has at least one absorption maximum in the range of 380 to 1200 nm, whereby for the case that A1 and/or A2 represents a dye molecule from the class of polymethine dyes, which has at least one absorption maximum in the range of 380 to 1200 nm, A1 is linked to the N-terminal amino group, and A2 is linked to an amino group of the amino acid lysine or to a hydroxy group of the amino acid serine in any position within the amino acid sequence (X)m, and their physiologically compatible salts.
Fragments, partial sequences, derivatives or analogs of the above-mentioned peptides stand, i.a., for shortened amino acid sequences, exchanges of individual or all amino acids for the corresponding D-amino acids, exchanges of individual amino acids for other amino acids, inverted sequences and combinations of the above-mentioned features.
The fragments, partial sequences, derivatives or analogs of the above-mentioned peptides can also contain amino acids that are not natural, such as, e.g., naphthalanine, cyclohexylalanine, norleucine, norvaline, xcex1-aminoadipic acid, xcex1-aminobutyric acid, xcex2-alanine, xcex2-cyclohexylalanine, ornithine, sarcosine or xcex4-hydroxylysine.